Rubella virus (RUBV) is a major human pathogen that causes birth defects and is associated with autoimmune disease. RUBV is a simple, plus-strand RNA virus (rubivirus genus, Togavirus family) that has five genes: two replicase genes and three structural protein genes that comprise the virus particle. We have made the unexpected discovery that one of the virion proteins, the capsid protein (C), is involved in an early step in virus RNA synthesis. Using replicons, constructs in which the structural genes have been replaced with a reporter gene, we have found that C enhances the rate of wild type replicon RNA accumulation and rescues deletions in three regions of the genome, at the 5'and 3'ends and internally within the gene of one of the replicase proteins. Our overarching hypothesis is that C executes or compensates for functions normally associated with the structure of the genomic RNA. In the proposed research, four Specific Aims will be pursued. In the first Specific Aim, we will test the hypothesis that C-mediated rescue of the internal deletion is through compensation for RNA structure rather than compensation for protein function. In the second Specific Aim, we will test the hypothesis that C binding to the genomic RNA is necessary for enhancement of wt replicon RNA replication and mutant rescue. In the third Specific Aim we will test whether C stabilizes replicon RNAs or serves as an RNA chaperone, both potential mechanisms by which C executes wt enhancement or mutant rescue. Finally, we have found that C associates with one of the replicase proteins, a novel finding among plus-strand RNA viruses, suggesting a model for involvement of C in viral RNA synthesis by which the C recruits both the viral RNA and the replicase proteins, facilitating formation of the replication complex and/or initiation of RNA synthesis. To test this model, the binding sites on C for the replicase protein will be mapped and mutants that fail to bind the replicase will be identified and used to see if they abrogate wt replicon enhancement and mutant rescue. We have evidence that the capsid protein of West Nile virus (Flavivirus family) enhances the replication of a West Nile virus replicon, indicating that C involvement in viral RNA synthesis that we are studying with RUBV may represent a paradigm among plus-strand RNA virus families. The function of capsid protein in viral RNA synthesis appears to at a step between penetration and unencapsidation of the virion and establishment of viral RNA replication complexes and thus the proposed research should yield insight into this poorly defined part of the virus replication cycle. PUBLIC HEALTH RELEVANCE: The proposed research will specifically explore how the capsid protein of rubella virus, a protein that makes up part of the virus particle, is also involved in viral RNA synthesis. Capsid proteins of other medically important viruses may play a similar role. A major hypothesis to be tested in the proposed research is that capsid protein involvement in viral RNA synthesis is through direct interaction of the capsid and replicase proteins and this interaction is an attractive target for antiviral drug development.